Nupage mes sds running buffer

Nuclear extract from 10 × 10⁶ mESCs was obtained by lysis in Buffer A (final: 25 mM Hepes pH 7.6, 5 mM MgCl₂, 25 mM KCl, 0.05 mM EDTA, 10% Glycerol, 1 mM DTT, 1 mM PMSF, 1× Complete Mini protease inhibitor) followed by collection in RIPA buffer (final: 150 mM NaCl, 1% triton, 0.5% sodium deoxy-cholate, 0.1% SDS, 50 mM Tris pH 8.0). Nuclear extracts were homogenized by sonication in a Diagenode Bioruptor and concentration was determined by Bradford assay (Biorad). 20 µg/lane total protein was run on Novex Life Technology NuPAGE 4–12% Bis-Tris gels in Invitrogen NuPAGE MES SDS Running Buffer and transferred on a Merck Chemicals Immobilon-P Membrane (PVDF 45 µm). The membrane was blocked (5% non-fat dry milk in 1× PBS, 0.1% Tween 20) and incubated in 5% non-fat dry milk in 1× PBS and 0.1% Tween 20 with the primary antibodies as listed in Supplementary Table 2. Finally, the membrane was incubated with corresponding secondary HRP coupled antibodies (5% non-fat dry milk in 1× PBS, 0.1% Tween 20), developed using Clarity Western ECL Substrate (Biorad) and imaged by a ChemiDoc XRS+ Imaging system (Biorad). For uncropped Western blots with molecular weight markers, please see Supplementary Figs. 2, 8, 10, 12 and 14.